PMC

Heat map of the 323 proteins significantly altered at one or more time points. Red and green rectangles represent significant up- or down- regulations, respectively, while the black color denotes that no significant change was found.

Efficient and reproducible extraction and sample preparation for the 60 animals. (A) Protein extraction yields from liver samples using a unique Polytron/sonication protocol with a cocktail of high-concentration of detergents. (B) The peptide recovery of the precipitation/on-pellet-digestion procedure. Peptide recovery was determined by a modified BCA method described previously.

Gene ontology annotation of significantly altered proteins. The distributions of the altered proteins by (A) cellular components and (B) biological processes. Representative time courses of the number of changed proteins are shown in panels (C)–(F). The up- and down- regulated proteins are denoted in red and green, respectively.

Experimental procedure for the large-scale, ion-current-based proteomic profiling. The ADX rats were dosed with 50 mg/kg methylprednisolone (MPL) (11 time-course groups) or saline (1 vehicle-control group). A multiplex, reproducible and extensive ion-current-based strategy was developed and evaluated for quantification in all animals. To determine the proper cutoff thresholds for discovery, the false altered protein discovery in each time-course group was estimated by comparison with a sham sample set (5 controls vs 5 controls).

Time series changes of hepatic metabolism. Panel (A) shows a simplified scheme for the hepatic metabolism pathways altered by MPL involving protein degradation, amino acid metabolism, and gluconeogenesis. The temporal profiles for the key proteins involved in amino acid metabolism, including cytosolic aspartate aminotransferase (cASAT), tyrosine aminotransferase (TAT), and alanine aminotransferase (AAT), are shown in panels (B) and (C). The profiles for key proteins involved in gluconeogenesis, pyruvate carboxylase (PC), phosphoenol pyruvate carboxykinase (PEPCK), and fructose 1,6-biphosphataseisozyme (FBPase) are shown in panel (D).

The extensive and reproducible separation of rat liver digests. (A) A representative base peak chromatogram for separation of liver digest. (B) Variation (RSD%) of the retention times of base peak peptides across 60 consecutive runs of a pooled liver digest, over a 18-day period. The nano-LC/MS configuration was shown in , and the separation was carried out on a 100-cm-long nano column (50-μm ID and packed with 2-μm C18 particles) heated at 52 °C. The extensive separation permits extensive analysis and accurate match of peptide peaks, which is critical for the ion-current-based quantification.